Mouse Model Of Colon Carcinoma Research Articles

Enhanced antitumour response of gold nanostar-mediated photothermal therapy in combination with immunotherapy in a mouse model of colon carcinoma.

This study investigated the potential of combining PTT with dendritic cell (DC)-based immunotherapy and anti-PD-L1 immune checkpoint blockade (ICB) therapy against colorectal cancer and elucidated the underlying mechanisms. The CT26 tumour-bearing mice were divided into seven treatment groups: control, atezolizumab (A), dendritic cells (DC), pAuNSs-mediated PTT (PTT), PTT combined with atezolizumab (PTT + A), PTT combined with dendritic cells (PTT + DC), and PTT combined with dendritic cells and atezolizumab (PTT + DC + A). Therapeutic efficacy was monitored. PTT upregulated most immune cell membrane receptor genes, including PD-L1, and downregulated genes associated with antigen presentation and T cell activation. Although the PTT + A and PTT + DC treatments showed partial tumour growth retardation, the combination of PTT with DCs and atezolizumab (PTT + DC + A) exhibited the most significant antitumour effect, with a complete remission rate of 50% and prolonged survival. On day 14, tumour samples from non-responsive mice revealed insufficient recruitment of T cells as the reason for uncured tumours. Notably, mice cured with PTT + DC and PTT + DC + A treatments showed no detectable lung nodules. This study demonstrated that the combination of PTT with DC-based immunotherapy and atezolizumab effectively overcomes the non-sensitive nature of CT26 tumours. These findings highlight the potential of this combination approach for colorectal cancer treatment.

Tumor Regression upon Intratumoral and Subcutaneous Dosing of the STING Agonist ALG-031048 in Mouse Efficacy Models.

Stimulator of interferon genes (STING) agonists have shown potent anti-tumor efficacy in various mouse tumor models and have the potential to overcome resistance to immune checkpoint inhibitors (ICI) by linking the innate and acquired immune systems. First-generation STING agonists are administered intratumorally; however, a systemic delivery route would greatly expand the clinical use of STING agonists. Biochemical and cell-based experiments, as well as syngeneic mouse efficacy models, were used to demonstrate the anti-tumoral activity of ALG-031048, a novel STING agonist. In vitro, ALG-031048 is highly stable in plasma and liver microsomes and is resistant to degradation via phosphodiesterases. The high stability in biological matrices translated to good cellular potency in a HEK 293 STING R232 reporter assay, efficient activation and maturation of primary human dendritic cells and monocytes, as well as long-lasting, antigen-specific anti-tumor activity in up to 90% of animals in the CT26 mouse colon carcinoma model. Significant reductions in tumor growth were observed in two syngeneic mouse tumor models following subcutaneous administration. Combinations of ALG-031048 and ICIs further enhanced the in vivo anti-tumor activity. This initial demonstration of anti-tumor activity after systemic administration of ALG-031048 warrants further investigation, while the combination of systemically administered ALG-031048 with ICIs offers an attractive approach to overcome key limitations of ICIs in the clinic.

The CX3CL1-CX3CR1 chemokine axis can contribute to tumor immune evasion and blockade with a novel CX3CR1 monoclonal antibody enhances response to anti-PD-1 immunotherapy.

CX3CL1 secreted in the tumor microenvironment serves as a chemoattractant playing a critical role in metastasis of CX3CR1 expressing cancer cells. CX3CR1 can be expressed in both cancer and immune-inhibitory myeloid cells to facilitate their migration. We generated a novel monoclonal antibody against mouse CX3CR1 that binds to CX3CR1 and blocks the CX3CL1-CX3CR1 interaction. We next explored the immune evasion strategies implemented by the CX3CL1-CX3CR1 axis and find that it initiates a resistance program in cancer cells that results in 1) facilitation of tumor cell migration, 2) secretion of soluble mediators to generate a pro-metastatic niche, 3) secretion of soluble mediators to attract myeloid populations, and 4) generation of tumor-inflammasome. The CX3CR1 monoclonal antibody reduces migration of tumor cells and decreases secretion of immune suppressive soluble mediators by tumor cells. In combination with anti-PD-1 immunotherapy, this CX3CR1 monoclonal antibody enhances survival in an immunocompetent mouse colon carcinoma model through a decrease in tumor-promoting myeloid populations. Thus, this axis is involved in the mechanisms of resistance to anti-PD-1 immunotherapy and the combination therapy can overcome a portion of the resistance mechanisms to anti-PD-1.

The effect of m2 peptide targeted nanoliposomes containing crocin on induction of phenotypic change in tumor macrophages to M1 state

AimsCrocin has immunomodulatory and anticancer effects. In this study, crocin was used to induce the M1 phenotype in mouse tumor macrophages. Main methodsA targeted liposomal formulation with m2 peptide was prepared and characterized to deliver crocin to the M2 macrophages present in the tumor environment. RT-qPCR and IHC were performed for in vitro and in vivo (in C26 colon carcinoma mouse model at a dose of 50 mg/kg) assessment of M1 induction, respectively. Key findingsIn vitro results indicated that liposome modified with m2 peptide was non-toxic to macrophages and had an improved uptake by macrophages compared to the non-targeted formulation and induced M1 phenotype through an IL6-independent pathway. M2 peptide- modified liposome showed considerable tumor accumulation and anti-tumor effects and significantly shifted the phenotype of tumor macrophages towards an anti-tumor M1 phenotype. SignificanceProbably the remarkable anti-tumor responses observed in this study with m2 peptide-targeted liposomal formulations containing crocin were due to the enhanced delivery of crocin to the tumor macrophage and the subsequent initiation of anti-tumor immune responses.

Effect of TFF2-MSA on tumor growth and survival in anti-PD-1 treated MC38 and CT26.wt colorectal cancer models by targeting MDSCs.

e15654 Background: Myeloid-derived suppressor cells (MDSCs) in the tumor microenvironment are potential therapeutic targets in immune checkpoint cancer therapy, particularly for cancers that are unresponsive to anti-PD-1 therapy. It has previously been demonstrated that trefoil factor family 2 (TFF2), a secreted anti-inflammatory peptide, can partially suppress MDSC expansion and partially activate tumor immunity through agonism of the CXCR4 receptor. We investigated whether a novel fusion protein, TFF2-murine serum albumin (TFF2-MSA), has single agent activity and can improve on the therapeutic effects of anti-PD-1 in the MC38 and CT26.wt syngeneic mouse models of advanced colorectal cancer (CRC). Methods: Two syngeneic colon carcinoma mouse models were developed using the MC38 and CT26.wt CRC cell lines grafted subcutaneously into C57BL/6 and BALB/C mice, respectively. We generated a recombinant fusion protein, designated TFF2-MSA, which contains murine TFF2 fused to murine serum albumin (MSA), for the purpose of increasing half-life and reducing the frequency of dosing. Mice subsequently received TFF2-MSA, anti-PD-1 antibody (clone 29F.1A12) or combination TFF2-MSA/anti-PD-1. Tumor volume, and survival were measured. At the endpoint, flow cytometry was performed on the draining and axillary lymph nodes, to examine treatment-induced effects on cellular immune profiles. Results: In the MC38 model, on Day 49 of treatment, tumor growth was suppressed (TGI) by TFF2-MSA alone, anti-PD-1 alone, and by combination TFF2-MSA/anti-PD-1 by 50%, 82% and 87% TGI, respectively. The survival rates at 50 days of mice treated with vehicle, TFF2-MSA, anti-PD-1, or combination TFF2-MSA/anti-PD-1 were 20%, 50% and 80%, and 90%, respectively. In the combination TFF2-MSA/anti-PD-1 treated mice, 40% demonstrated a complete response. In the CT26.wt model, tumor growth was suppressed by TFF2-MSA, anti-PD-1 and by combination TFF2-MSA/anti-PD-1 by 17%, 43% and 67%, respectively. Survival in the CT26.wt model at Day 30 treated with vehicle, TFF2-MSA, anti-PD1 and the combination of TFF2-MSA/anti-PD-1 was 0%, 40%, 60% and 60%, respectively. The percentage of exhausted CD8+ T cells was markedly reduced in the draining lymph node by the combination TFF2-MSA/anti-PD-1 treatment, as measured by flow cytometry using antibodies against LAG3, TIM3, and PD-1. Conclusions: TFF2-MSA has single agent activity and is additive to anti-PD-1 antibody checkpoint inhibition in treating syngeneic mouse models of advanced CRC. In a separate abstract, additive effects between TFF2-MSA and anti-PD-1 antibody were also demonstrated in a separate ACKP (Atp4b-Cre; Cdh1-/-; LSL-KrasG12D; Trp53-/-) gastric cancer model, suggesting combination therapy may also be applicable to gastric cancer.

Abstract 6800: Unique autologous cancer vaccine comprised of irradiated whole tumor cells and MBTA (rWTC-MBTA) triggers antitumor immune response to prevent metastasis

Abstract Despite the advances made in cancer treatment over the past decades, one statistic has remained unchanged: metastatic cancer accounts for 90 percent of annual cancer deaths in the United States. Our group previously demonstrated that an autologous whole tumor cell vaccine (rWTC-MBTA: irradiated autologous whole tumor cells pulsed with Mannan-BAM, TLR ligands, and anti-CD40 antibody) had a potent anti-tumor immune response and prolonged survival in a mouse colon carcinoma model. To investigate whether rWTC-MBTA would work in “cold” tumor models, we evaluated the vaccine’s effect on preventing and treating tumor metastasis in 4T1 breast tumors. Our data showed that vaccinated mice could significantly prevent lung metastasis in both intravenous injection and mammary pad subcutaneous implantation animal models. Further, we used another metastasis model that more closely mimics clinical practice by resecting the primary tumor after metastasis development. The data showed that vaccinated mice could prevent tumor recurrence, increase T-cell infiltration in the metastatic tumor, and prolong the survival curve. The mechanistic investigation by immunophenotyping revealed that rWTC-MBTA vaccination induced both effector memory (CD44+CD62L−) and center memory (CD44+CD62L+) T cells as well as increased overall CD4+ and CD8+ T-cell count while depletion experiments demonstrated that CD8+ T cells were required for vaccine efficacy. Isolated splenocytes co-cultured with 4T1 cells showed rWTC-MBTA significantly increased T-cell mediated cytotoxicity through TNF-a and IFN-γ in CD107a+CD4+ T cells and Granzyme B and IFN-γ in CD107a+CD8+ T cells. In all experiments, vaccination exerted negligible systemic toxicity. Collectively, our study demonstrates that the rWTC-MBTA vaccine is a safe and promising therapeutic option to prevent and treat tumor metastasis by triggering an antitumor immune response. Citation Format: Juan Ye, Herui Wang, Mitchell Sun, Ondrej Uher, Mark R. Gilbert, Karel Pacak, Zhengping Zhuang, Rogelio Medina, Samik Chakraborty, Jan Zenka. Unique autologous cancer vaccine comprised of irradiated whole tumor cells and MBTA (rWTC-MBTA) triggers antitumor immune response to prevent metastasis. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6800.

Abstract 704: MDSC-targeted TFF2-MSA suppresses tumor growth and increases survival in anti-PD-1 treated MC38 and CT26.wt murine colorectal cancer models

Abstract Aims: Myeloid-derived suppressor cells (MDSCs) in the tumor microenvironment are a potential therapeutic target in immune checkpoint cancer therapy, but MDSC-targeted therapies have yet been shown to improve survival. Trefoil factor family 2 (TFF2), a secreted anti-inflammatory peptide, can suppress MDSC expansion and activate tumor immunity in part through agonism of the CXCR4 receptor. The aim of this study is to investigate whether a novel TFF2 - albumin fusion peptide (TFF2-MSA) can improve survival in anti-PD-1 treated syngeneic colorectal cancer (CRC) mouse models. Methods: Two syngeneic colon carcinoma mouse models were developed using cell lines grafted subcutaneously into mice. MC38 CRC cells were engrafted into C57BL/6 mice while CT26.wt CRC cells were implanted into BALB/C mice We generated a recombinant fusion protein, designated TFF2-MSA, which contains murine TFF2 fused to murine serum albumin (MSA), for the purpose of increasing half-life and reducing dose frequency. Mice subsequently received either TFF2-MSA or anti-PD-1 antibody (clone 29F.1A12) or both, and tumor volume, and survival were measured. At the endpoint, flow cytometry was performed to examine treatment-induced effects on immune profiles. Results: In the MC38 model, administration of TFF2-MSA suppressed tumor growth (TGI 38%), the combination of TFF2-MSA and anti-PD-1 had an additive effect and suppressed tumor growth dramatically (TGI 74%). The combination also exhibited a survival rate of 90% after 50 days, while vehicle and single TFF2-MSA therapy were 30% and 65%, respectively. The percentage of exhausted CD8+ T cells was markedly reduced in the draining lymph node by the combination treatment, as measured by flow cytometry using antibodies against LAG3, TIM3 and PD-1. In the CT26.wt model, administration of TFF2-MSA alone exhibited little effect, but the combination of anti-PD-1 and TFF2-MSA showed a profound effect. In the CT26.wt model, administration of TFF2-MSA suppressed tumor growth (TGI 17%), anti-PD-1 alone (TGI 43%) and the combination of TFF2-MSA and anti-PD-1 (TGI 54%). Conclusion: Targeting MDSCs using TFF2-MSA fusion protein synergizes well with PD-1 blockade therapy in advanced and metastatic syngeneic mouse models of colorectal cancer. In a separate abstract, additive effects between TFF2-MSA and anti-PD-1 antibody were also demonstrated in a separate ACKP (Atp4b-Cre; Cdh1-/-; LSL-KrasG12D; Trp53-/-) gastric cancer model, suggesting combination therapy may also be applicable to gastric cancer. Citation Format: Bruce L. Daugherty. MDSC-targeted TFF2-MSA suppresses tumor growth and increases survival in anti-PD-1 treated MC38 and CT26.wt murine colorectal cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 704.

Peptide-Based Cancer Vaccine Delivery via the STINGΔTM-cGAMP Complex.

With the advent of bioinformatic tools in efficiently predicting neo-antigens, peptide vaccines have gained tremendous attention in cancer immunotherapy. However, the delivery of peptide vaccines remains a major challenge, primarily due to ineffective transport to lymph nodes and low immunogenicity. Here, a strategy for peptide vaccine delivery is reported by first fusing the peptide to the cytosolic domain of the stimulator of interferon genes protein (STINGΔTM), then complexing the peptide-STINGΔTM protein with STING agonist 2'3' cyclic guanosine monophosphate-adenosine monophosphate (cGAMP). The process results in the formation of self-assembled cGAMP-peptide-STINGΔTM tetramers, which enables efficient lymphatic trafficking of the peptide. Moreover, the cGAMP-STINGΔTM complex acts not only as a protein carrier for the peptide, but also as a potent adjuvant capable of triggering STING signaling independent of endogenous STING protein-an especially important attribute considering that certain cancer cells epigenetically silence their endogenous STING expression. With model antigen SIINFEKL, it is demonstrated that the platform elicits effective STING signaling in vitro, draining lymph node targeting in vivo, effective T cell priming in vivo as well as antitumoral immune response in a mouse colon carcinoma model, providing a versatile solution to the challenges faced in peptide vaccine delivery.

The Beneficial Effect of IL-12 and IL-18 Transduced Dendritic Cells Stimulated with Tumor Antigens on Generation of an Antitumor Response in a Mouse Colon Carcinoma Model

The main purpose of our study was to determine the effect of dendritic cell (DC) transduction with lentiviral vectors carrying sequences of il18 and/or il12 genes on the level of antitumor activity in vitro and in vivo. We examined the ability of DCs to migrate to the tumor-draining lymph nodes and infiltrate tumor tissue and to activate the local and systemic antitumor response. On the 15th day, DCs genetically modified for production of IL-12 and/or IL-18 were administered peritumorally to C57BL/6 female mice with established MC38 tumors. Lymphoid organs and tumor tissue were collected from mice on the 3rd, 5th, and 7th days after a single administration of DCs for further analysis. Administration of DCs transduced for production of IL-12 alone and in combination with IL-18 increased the inflow and activity of CD4+ and CD8+ T lymphocytes in the tumor microenvironment and tumor-draining lymph nodes. We also found that even a single administration of such modified DCs could trigger a systemic antitumor response as well as inhibit tumor growth. Application of the developed DC-based vaccines may exert a favorable impact on stimulation of an antitumor immune response, especially if these DC vaccines are administered repeatedly.

Deletion of IP6K1 in mice accelerates tumor growth by dysregulating the tumor-immune microenvironment

ABSTRACT A family of inositol hexakisphosphate kinases (IP6Ks) catalyzes the production of inositol pyrophosphate IP7 (5-diphosphoinositolpentakisphosphate) which is known to modulate various biological events such as cell growth. While targeting IP6K1 in various cancer cells has been well reported to control cancer cell motility and invasiveness, the role of host IP6K1 in tumor progression remains unknown. By using a syngeneic MC38 murine mouse colon carcinoma model, here we examined how host IP6K1 in the tumor microenvironment influences tumor growth. In IP6K1 knockout (KO) mice, the growth of MC38 tumor cells was markedly accelerated and host survival was significantly shortened compared with wild-type (WT). Our flow cytometric analysis revealed that tumors grown in IP6K1 KO mice had lower immune suppressive myeloid cells and M1 polarized macrophages. Notably, infiltration of both antigen-presenting dendritic cells and CD8+ cytotoxic T lymphocytes into the tumor tissues was remarkably abrogated in IP6K1 KO condition. These studies suggest that enhanced tumor growth in IP6K1 KO mice resulted from reduced anti-tumor immunity due to disturbed immune cell actions in the tumor microenvironment. In conclusion, we demonstrate that host IP6K1 acts as a tumor suppressor, most likely by fine-tuning diverse tumor-immune cell interactions, which might have implications for improving the host response against cancer progression.

Synergistic effect of radiotherapy and dendritic-cell-based immunotherapy via miR-15b-5p regulation in a colon carcinoma mouse model

Purpose: To determine the combined effect of radiotherapy (RT) and dendritic cell (DC)-based immunotherapy on tumor regression, and the influence of miR-15b-5p on the sensitivity of the tumor to RT/DC.
 Methods: Colon carcinoma cell line (T-26) was injected subcutaneously into BALB/c mice to generate xenograft tumors. The animals were exposed to radiation therapy (RT) only or DC only, or RT plus DC (RT/DC). Tumor progression was measured and analyzed followed by quantification of miR-15b-5p using qPCR in each group 21 days after tumor inoculation. Overexpressed xenograft tumors were generated by transfecting MiR-15b-5p mimic to CT-26 cell lines and subcutaneously administered to mice. The mice were then subjected to RT/DC treatment and assessed for tumor growth, TUNEL staining, as well as mRNA and protein expressions of B-cell lymphoma-2 (BCL-2), caspase-3 (casp-3), and X-linked inhibitor of apoptosis protein (XIAP).
 Results: RT/DC combination treatment resulted in tumor regression and increased miR-15b-5p expression, when compared with other groups treated with RT and DC alone (p < 0.001). There was a marked reduction in tumor growth following RT/DC combination therapy in miR-15b-5p overexpressed xenograft tumor (p < 0.001). Moreover, miR-15b-5p/(RT/DC) treatment significantly increased cancer cell apoptosis. These results were substantiated by increased mRNA and protein expressions of casp-3, reduced BCL-2 and XIAP (p < 0.001).
 Conclusion: These findings demonstrate that RT/DC combination therapy increases miR-15b-5p levels in mice, and that miR-15b-5p upregulation enhances the sensitivity of tumors to RT/DC combination therapy. Therefore, the combination of RT and dendritic cell-based immunotherapy offers significant therapeutic advantage in controlling cancers, including metastatic tumors.

Abstract 4520: Preclinical characterization of ALG-031048, a novel STING agonist with potent anti-tumor activity in mice

Abstract Introduction: The STING (STimulator of INterferon Genes) protein has been identified as an attractive target for cancer immunotherapy due to its central role in inducing T-cell mediated tumor control. Here we describe the preclinical characteristics of the novel STING agonist, ALG-031048, that demonstrates potent anti-tumoral activity in in vivo mouse models. Methods: Binding to STING was determined using differential scanning fluorimetry (DSF). Cellular activation and cytokine release were assessed using HEK 293T reporter cell lines as well as THP-1 and Raw264.7 cells. In vitro stability was measured in the presence of snake venom phosphodiesterase (SVPD). In vivo anti-tumoral activity was tested in the syngeneic CT26 and B16F10 mouse model. Results: ALG-031048 demonstrated potent binding to the STING R232 protein in the DSF assay with a mean Kd of 3.12 ± 0.12 μM and a thermal shift of 12.8 °C, similar to the natural ligand 2'3'cGAMP (Kd of 2.75 ± 0.78 μM, shift of 16.7°C) or the clinical stage STING agonist, ADU-S100 (Kd of 4.61 ± 0.42 μM, shift of 10.7°C). In the HEK 293T R232 reporter assay, ALG-031048 was more potent than ADU-S100 with a mean EC50 of 55.9 ± 24.1 and 30.3 ± 17.1 nM for the IFNβ and IRF reporter, respectively, compared to ADU-S100 with EC50 values of 233 ± 179 and 88.7 ± 35.6 nM. Cellular activation was confirmed in THP-1 and Raw264.7 cells where ALG-031048 induced the secretion of IFNβ and CXCL-10. ALG-031048 was highly stable and exhibited no degradation for up to 24 hours in the presence of SVPD while ADU-S100 and 2'3'cGAMP were quickly degraded with less than 10% remaining after 24 hours. In vivo, 3 intra-tumoral (IT) doses of 100 μg ALG-031048 q3d caused complete tumor regression in 90% of animals in the CT26 mouse colon carcinoma model, while treatment with 25 μg ALG-031048 IT 3xq3d resulted in survival of 60 % of animals. In contrast, only 10 and 44% of mice showed complete tumor regression when treated IT 3xq3d with 25 or 100 μg ADU-S100, respectively. To study whether induction of a protective immune response was associated with STING agonist treatment, surviving animals were re-challenged with a second inoculation of CT26 cells on the contra-lateral side. All naïve animals formed tumors within 23-30 days. In contrast, 5 out of 5 and 8 out of 9 mice pre-treated with 25 or 100 μg ALG-031048, respectively, did not exhibit any significant tumor growth over 40 days, while 3 out of 4 animals pretreated with 100 μg ADU-S100 showed complete tumor suppression. Initial results using the B16F10 mouse melanoma model confirmed the potent anti-tumoral activity of ALG-031048 observed in the CT26 model. Summary: ALG-031048 is a novel STING agonist combining high stability and excellent in vitro potency. In preclinical models, ALG-031048 demonstrated potent and long-lasting tumor suppression. ALG-031048 is advancing in toxicology studies for further profiling as a clinical candidate. Citation Format: Andreas Jekle, Santosh Thatikonda, Sarah Stevens, Caroline Williams, April Kinkade, Suping Ren, Ruchika Jaisinghani, Qingling Zhang, Dinah Misner, Antitsa Stoycheva, Jerome Deval, Sucheta Mukherjee, Francois Gonzalvez, Sushmita Chanda, David B. Smith, Julian A. Symons, Lawrence M. Blatt, Leonid Beigelman. Preclinical characterization of ALG-031048, a novel STING agonist with potent anti-tumor activity in mice [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4520.

Abstract A32: The SUV39H1-H3K9me3 pathway represses cytotoxic T lymphocyte effector expression to confer colon carcinoma immune escape

Abstract Despite the presence of cytotoxic T lymphocytes (CTLs) in the tumor microenvironment, the majority of immunogenic human colon cancers do not respond to immune checkpoint inhibitor immunotherapy and microsatellite instable (MSI) tumors are not naturally eliminated. The molecular mechanism underlying the inactivity of tumor-infiltrating CTLs is unknown. We report here that CTLs are present in both MSI and microsatellite stable (MSS) colon tumors. The expression of the H3K9me3-specific histone methyltransferase SUV39H1 is significantly elevated in human colon carcinoma as compared to normal colon tissues. Using a mouse colon carcinoma model, we further determined that tumor-infiltrating CTLs in the colon tumor microenvironment express high levels of SUV39H1. To target SUV39H1 in the tumor microenvironment, a virtual chemical library was screened based on the SET domain structure of human SUV39H1 protein. Functional enzymatic activity assays identified a small molecule that inhibits SUV39H1 enzymatic activity. Based on the structure of this small molecule, we modified it and chemically synthesized a novel small molecule, termed F5446, which has an EC50 of 0.496 μM for SUV39H1 enzymatic activity. H3K9me3 is enriched in the promoters of GZMB, PRF1, FASLG and IFNG in quiescent T cells. F5446 inhibits H3K9me3, thereby upregulating expression of these effectors in tumor-infiltrating CTLs and suppressing colon carcinoma growth in a CD8+ CTL-dependent manner in vivo. Our data indicate that SUV39H1 represses CTL effector expression and, in doing so, confers colon cancer immune escape. Citation Format: Chunwan Lu, Dafeng Yang, John D. Klement, II Kyu Oh, Natasha M. Savage, Jennifer L. Waller, Aaron H. Colby, Mark W. Grinstaff, Nicholas H. Oberlies, Cedric Pearce, Zhiliang Xie, Samuel Kulp, Christopher Coss, Mitch A. Phelps, Thomas Albers, Iryna O. Lebedyeva, Kebin Liu. The SUV39H1-H3K9me3 pathway represses cytotoxic T lymphocyte effector expression to confer colon carcinoma immune escape [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A32.

Use of a genetically engineered E. coli overexpressing β-glucuronidase accompanied by glycyrrhizic acid, a natural and anti-inflammatory agent, for directed treatment of colon carcinoma in a mouse model.

Bacteria-directed enzyme prodrug therapy (BDEPT), is an emerging alternative directed and tumor-specific approach. The basis of this method is the conversion of a non-toxic prodrug by a bacterial enzyme to a toxic drug within the tumor-microenvironment (TME). In the present study, the therapeutic efficacy of BDEPT was investigated based on the ability of Escherichia coli DH5α-lux/βG in activation of glycyrrhizic acid (GL), a natural and non-toxic compound purified from licorice, to glycyrrhetinic acid (GA) only in TME. To do so, the anti-bacterial effects of GL on bacteria and the cytotoxic effects of the produced GA on survival rate of CT26 mouse colon carcinoma cells were evaluated. The IC50 values of the produced GA and cisplatin were determined as 210μM and 100μM, respectively. Comparing these values to GL treatment (1305μM) indicates that bacteria could have efficiently activated GL to GA to inhibit the growth of tumor cells. Afterward, the anti-cancer effects of bacteria used in combination with GL was investigated in a mouse model of colon carcinoma. Results were indicative of targeted homing and even proliferation of luminescent bacteria in TME. Moreover, combined treatment greatly inhibited tumor growth. Histopathological analysis of dissected tissues also demonstrated increased apoptosis rate in tumor cells after combined treatment and interestingly, showed no obvious damage to the spleen and liver of treated mice. Accordingly, this BDEPT approach could be considered as an effective alternative tumor-specific therapy utilizing prodrug-activating enzymes expressing from tumor-targeting bacteria to allow the development of new tumor-specific pharmacotherapy protocols.

Cell Wall Membrane Fraction of Chlorella sorokiniana Enhances Host Antitumor Immunity and Inhibits Colon Carcinoma Growth in Mice.

A colon cancer growth inhibitor partially purified from the isolated cell wall membrane fraction of Chlorella sorokiniana, here referred to as Chlorella membrane factor (CMF), was evaluated for its antitumor and immunomodulatory effects in cell culture and in a colon carcinoma mouse model. The CMF treatment dose- and time-dependently inhibited colon carcinoma cell growth in 2-dimensional cultures. Treatment with CMF also significantly inhibited the growth of colon carcinoma spheroids in 3-dimensional cell culture in coculture with T lymphocytes. In a mouse CT26 colon carcinoma peritoneal dissemination model, intraperitoneal injection of CMF (10 or 30 mg dry weight/kg body weight, every other day) dose-dependently and significantly attenuated the growth of tumor nodules via induction of tumor cell apoptosis. Evaluation of immune cell populations in ascites showed that CMF treatment tended to increase T lymphocytes but lower granulocyte populations. The present study suggests that the cell wall membrane fraction of Chlorella sorokiniana contains a bioactive material that inhibits colon carcinoma growth via direct cell growth inhibition and stimulation of host antitumor immunity. Hence, it is suggested that the Chlorella cell wall membrane extract or a bioactive substance in the extract is an attractive complementary medicine for cancer therapy.

Targeting of DDR1 with antibody-drug conjugates has antitumor effects in a mouse model of colon carcinoma.

DDR1 has been identified as a cancer‐associated receptor tyrosine kinase that is highly expressed in several malignancies relative to normal tissues. Clinically approved multi‐kinase inhibitors, such as nilotinib, inhibit DDR1‐mediated tumor growth in xenograft models, suggesting DDR1 might be a potential target for cancer treatments. Here, we employed an antibody‐based strategy with a novel anti‐DDR1 antibody‐drug conjugate (ADC) for colon carcinoma treatment. We developed T4H11‐DM4, an ADC targeting DDR1 which carries the tubulin inhibitor payload DM4. Immunohistochemical analysis of a tissue microarray containing 100 colon cancer specimens revealed that DDR1 was highly expressed in 81% of tumor tissues. Meanwhile, high expression of DDR1 was associated with poor survival in patients. In vitro, T4H11‐DM4 exhibited potent anti‐proliferative activity with half maximal inhibitory concentration (IC50) values in the nanomolar range in a panel of colon cancer cell lines. In vivo, the antitumor efficacy of T4H11‐DM4 was evaluated in three colon cancer cell lines expressing different levels of DDR1. T4H11‐DM4 achieved complete tumor regression at doses of 5 and 10 mg·kg−1 in HT‐29 and HCT116 tumor models. Moreover, a correlation between in vivo efficacy of T4H11‐DM4 and the levels of DDR1 expression on the cell surface was observed. Tumor cell proliferation was caused by the induction of mitotic arrest, indicating that the antitumor effect in vivo was mediated by DM4. In addition, T4H11‐DM4 was efficacious in oxaliplatin‐resistant colon cancer models. In exploratory safety studies, T4H11‐DM4 exhibited no overt toxicities when multi‐doses were administered at 10 mg·kg−1 into BALB/c nude mice or when a single dose up to 50 mg·kg−1 was administered into BALB/c mice. Overall, our findings highlight the potential of DDR1‐targeted ADC and may facilitate the development of a new effective therapeutic strategy for colon cancer.

Abstract 4588: Characterization of anti-CTLA-4 effects on tumor growth and immune microenvironment in a syngeneic CT26.WT colon carcinoma mouse model

Abstract Colorectal cancer is the third most common type of cancer and one of the leading causes of cancer related deaths. It is a group of various diseases at molecular level, which can make targeted drug development challenging. However, novel therapeutics including immunotherapies have shown their potential to be utilized in the future also when treating colorectal cancer. One promising example is ipilimumab, a monoclonal antibody targeting CTLA-4 (cytotoxic T lymphocyte antigen-4, also known as CD152). CTLA-4 is a cell surface receptor expressed on activated T and B lymphocytes. It regulates immunological tolerance and downregulates immune responses. The aim of the study was to characterize the effects of anti-CTLA-4 on tumor growth, survival and tumor immune microenvironment of mice subcutaneously inoculated with mouse colon carcinoma CT26.WT cells. CT26.WT cells were subcutaneously inoculated into female Balb/c mice (n=27). Body weights and tumor dimensions were determined two times a week during the course of the study. The mice were randomized to three groups (n=9/group) with similar tumor volume and variation and dosing was started when an average tumor size of approximately 50 mm³ was reached (day 7). Anti-CTLA-4 antibody (9D9, 10 mg/kg), an isotype control (mouse IgG2b) and vehicle (PBS) were administered (i.p.) twice a week. Tumor growth and survival were followed for 26 days after inoculation of the cancer cells. The mice were sacrificed individually when the maximum tumor volume (1500 mm³) was reached, the tumors were ulcerated or at the latest on study day 26. Tumor samples were collected for histological and immunohistochemical analysis and stained for helper T cells (CD4), cytotoxic T cells (CD8), myeloid-derived suppressor cells (MDSC; CD11b), tumor-associated macrophages (F4/80), and inflammatory monocytes and neutrophils (FCGR1/CD64) for characterization of tumor immune microenvironment. Maximum tumor volume and tumor ulceration were observed the earliest on study day 17. Anti-CTLA-4 suppressed tumor growth, and all mice in the group receiving anti-CTLA-4 were still in the study on day 26. Only one and four mice were left in the vehicle and isotype control groups, respectively. Immunohistochemical staining suggested an increase of CD4+ tumor-infiltrating lymphocytes (TILs) in anti-CTLA-4 treated mice. Furthermore, more TAMs were observed in tumors of anti-CTLA-4 treated mice, especially in the borderlines of necrotic areas. Large quantity of CD11b+ MDSCs and inflammatory cells were observed in CT26.WT tumors. CT26.WT subcutaneous model can be utilized for studying the effects of immunotherapies, and anti-CTLA-4 treatment suppressed tumor growth in this model. CT26.WT tumors represent a highly immunogenic hot tumor type with high immune cell infiltration, associated with a good efficacy of anti-CTLA-4 treatment in the model. Citation Format: Jenni H. Mäki-Jouppila, Tiina E. Kähkönen, Mari I. Suominen, Jussi M. Halleen, Jenni Bernoulli. Characterization of anti-CTLA-4 effects on tumor growth and immune microenvironment in a syngeneic CT26.WT colon carcinoma mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4588.

Abstract 4144: Water extract from Chlorella vulgaris cell membrane fraction enhances host antitumor immune responses and inhibit colon carcinoma growth in mice

Abstract Natural products obtained from various resources are often used as new compounds or seed compounds of effective drugs for disease including cancer. The water extract partially purified from the cell membrane fraction of Chlorella vulgaris (CMWE) was evaluated on its antitumor and immunomodulatory effects in cell culture and a colon carcinoma mouse model. The CMWE was filtered through 0.22 μm pore size membrane for all experiments. In two-dimensional cell cultures, the CMWE treatment inhibited cell growth of both murine and human colon carcinoma cells in a dose- and time-dependent manner. In contrast, the cell proliferation of mouse splenocytes (SPLs) and bone marrow cells (BMCs) were stimulated by the treatment with CMWE. The treatment with CMWE also increased specific subpopulations of the cells in BMCs: antigen presenting cells (CD19+ B cells, 33D1+ dendritic cells and CD68+ macrophage), CD4+ and CD8+ T cells, whereas LY-6G+neutrophils were decreased. In a three-dimensional spheroid culture, spheroid growth of CT26 cells co-cultured with Jurkat cells (human T lymphoblasts) was significantly attenuated by CMWE treatment via induction of apoptosis. In a two-dimensional cell culture of Jurkat cells, mRNA levels of TNFα, IFNγ and granzyme B were significantly increased by CMWE treatment. In a mouse CT26 colon carcinoma peritoneal dissemination model, intraperitoneal injection of CMWE (30 mg/kg/day, started from 3 days after CT26 inoculation for 9 days, every other day) significantly attenuated the growth of CT26 colon carcinoma nodules in syngeneic mice by CMWE treatment-associated apoptosis without any noticeable adverse effect. Analysis of subpopulation of intraperitoneally infiltrating immune cells collected from CT26 cell tumor-bearing mice, indicated that CMWE treatment increased CD4+ and CD8+ T lymphocytes, CD19+ B cell, and CD68+ macrophage populations. In contrast, LY6G+neutrophil population was decreased as compared to PBS-treated group. The present study suggests that CMWE inhibits colon carcinoma growth via direct cell growth inhibition and a stimulation of the host antitumor immune responses. Taken together, the current study suggests that water extract obtained from the cell membrane fraction of Chlorella vulgaris contain significant bioactive materials that inhibit colon carcinoma growth. This study was supported by 2016EUGLENA-RC1 (MT), 2017EUGLENA-RC2 (MT), Kansas State University Johnson Cancer Research Center 2018CRA (NR and MT), and NIH grant P20 RR017686 (MT). Citation Format: Susumu Ishiguro, Nicole Robben, Deepa Upreti, Paige Cote, Sarah Greenway, Riley Burghart, Ayaka Nakashima, Masaaki Tamura. Water extract from Chlorella vulgaris cell membrane fraction enhances host antitumor immune responses and inhibit colon carcinoma growth in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4144.

SUV39H1 Represses the Expression of Cytotoxic T-Lymphocyte Effector Genes to Promote Colon Tumor Immune Evasion

Despite the presence of CTLs in the tumor microenvironment, the majority of immunogenic human colon cancer does not respond to immune checkpoint inhibitor immunotherapy, and microsatellite instable (MSI) tumors are not naturally eliminated. The molecular mechanism underlying the inactivity of tumor-infiltrating CTLs is unknown. We report here that CTLs were present in both MSI and microsatellite stable colon tumors. The expression of the H3K9me3-specific histone methyltransferase SUV39H1 was significantly elevated in human colon carcinoma compared with normal colon tissues. Using a mouse colon carcinoma model, we further determined that tumor-infiltrating CTLs in the colon tumor microenvironment have high expression of SUV39H1. To target SUV39H1 in the tumor microenvironment, a virtual chemical library was screened on the basis of the SET (suppressor of variegation 3-9, enhancer of zeste and trithorax) domain structure of the human SUV39H1 protein. Functional enzymatic activity assays identified a small molecule that inhibits SUV39H1 enzymatic activity. On the basis of the structure of this small molecule, we modified it and chemically synthesized a small molecule, termed F5446, which has an EC50 of 0.496 μmol/L for SUV39H1 enzymatic activity. H3K9me3 was enriched in the promoters of GZMB, PRF1, FASLG, and IFNG in quiescent T cells. F5446 inhibited H3K9me3, thereby upregulating expression of these effectors in tumor-infiltrating CTLs and suppressing colon carcinoma growth in a CD8+ CTL-dependent manner in vivo Our data indicate that SUV39H1 represses CTL effector gene expression and, in doing so, confers colon cancer immune escape.

Cancer-Stimulated CAFs Enhance Monocyte Differentiation and Protumoral TAM Activation via IL6 and GM-CSF Secretion.

Purpose: M2-type TAMs are increasingly implicated as a crucial factor promoting metastasis. Numerous cell types dictate monocyte differentiation into M2 TAMs via a complex network of cytokine-based communication. Elucidating critical pathways in this network can provide new targets for inhibiting metastasis. In this study, we focused on cancer cells, CAFs, and monocytes as a major node in this network.Experimental Design: Monocyte cocultures with cancer-stimulated CAFs were used to investigate differentiation into M2-like TAMs. Cytokine array analyses were employed to discover the CAF-derived regulators of differentiation. These regulators were validated in primary CAFs and bone marrow-derived monocytes. Orthotopic, syngeneic colon carcinoma models using cotransplanted CAFs were established to observe effects on tumor growth and metastasis. To confirm a correlation with clinical evidence, meta-analyses were employed using the Oncomine database.Results: Our coculture studies identify IL6 and GM-CSF as the pivotal signals released from cancer cell-activated CAFs that cooperate to induce monocyte differentiation into M2-like TAMs. In orthotopic, syngeneic colon carcinoma mouse models, cotransplanted CAFs elevated IL6 and GM-CSF levels, TAM infiltration, and metastasis. These pathologic effects were dramatically reversed by joint IL6 and GM-CSF blockade. A positive correlation between GM-CSF and IL6 expression and disease course was observed by meta-analyses of the clinical data.Conclusions: Our studies indicate a significant reappraisal of the role of IL6 and GM-CSF in metastasis and implicate CAFs as the "henchmen" for cancer cells in producing an immunosuppressive tumor ecological niche. Dual targeting of GM-CSF and IL6 is a promising new approach for inhibiting metastasis. Clin Cancer Res; 24(21); 5407-21. ©2018 AACR.